Staphylococcus aureus (SA) nasal carriage is a common condition in humans that acts as an important reservoir for spreading this pathogen, which causes more deaths each year in the U.S. than HIV/AIDS. Using antibiotics to decolonize the nose of SA, prior to hospitalization, significantly reduces the incidence of nosocomial infection, strongly suggesting that reducing or eliminating nasal carriage of SA would directly lead to a reduction in SA clinical infections. Topical antibiotics such as mupirocin can effectivel rid SA nasal carriage, but repetitive use leads to bacterial resistance. We are proposing a novel approach to reduce nasal carriage of SA, by improving upon the nose's natural immunity against SA. To accomplish this goal, we will restore the production of cyclic antimicrobial peptides called retrocyclins, which have been evolutionarily silenced in the human genome. While some nonhuman primates produce retrocyclin-like peptides, human retrocyclin genes harbor a premature termination codon that blocks translation of these peptides. Based on the genetic information contained within the otherwise intact retrocyclin genes, we first recreated retrocyclins synthetically, and discovered that retrocyclin peptides are potently active against methicillin-resistant SA (MRSA) strains and all other SA isolates tested, and can work in concert with at least one precursor peptide involved in retrocyclin biosynthesis. In related studies, we restored the natural production of retrocyclin peptides in cervicovaginal cells and tissues using aminoglycosides, which suppressed retrocyclin's premature termination codon, resulting in natural retrocyclin peptides that could protect against HIV infection. We now propose to correct retrocyclin deficiency in the nasal mucosa using a next- generation, non-antibiotic, read-through agent called PTC124, which pilot immunocytochemical studies indicate can produce retrocyclins in nasal epithelia. Based on our preliminary studies, we hypothesize that PTC124 will restore the natural production of retrocyclins in the nasal mucosa, and the precursor and mature forms will work in concert to confer sustained protection against SA colonization. Aim 1 will assess the ability of PTC124 to induce anti-SA retrocyclins in human nasal epithelia. We will confirm that human nasal epithelia, treated with PTC124, express intact retrocyclin peptides active against SA, determine the concentration of retrocyclins that is sufficient to protect nasal mucosa from SA colonization, and examine how long a single treatment of PTC124 can maintain retrocyclin-mediated protection. We will then explore which mature and precursor retrocyclins are produced, and where they reside subcellularly and extracellularly. Although there is considerable evidence that mechanisms underlying premature and normal codon termination differ substantially, and suppressing agents should not target normal termination codons, Aim 2 will verify that PTC124 does not induce off-target effects to nasal epithelia. Using agents such as PTC124 to bolster nasal innate immunity would likely reduce the incidence of nasal carriage, and subsequent transmission and infection by SA.